Diversity, phylogeny, and bathymetric zonation of Sirsoe (Annelida: Hesionidae) from colonization experiments in the South China Sea, with the description of three new species

Abstract The South China Sea (SCS) basin is hypothesized to host distinct and bathymetrically differentiated fauna due to its semi‐enclosed basin and three‐layer circulation system. To test this hypothesis, three cow falls are artificially deployed at separate depths (655, 1604, and 3402 m) on Zhongnan seamount in the middle SCS, and the associated worms, Sirsoe spp. are selected as targets to explore their diversity, phylogeny, and zonation pattern. Analyses of collected specimens reveal three new Sirsoe species, which were then nominally described and named as S. polita sp. nov. (655 m), S. nanhaiensis sp. nov. (1604 and 3402 m), and S. feitiana sp. nov. (3402 m), and one known species (S. balaenophila lineage II). Metabarcoding analyses on cow‐fall sediments reveal seven additional Operated Taxonomic Units (OTUs) assigned to Sirsoe, increasing the Sirsoe diversity to 10 species/OTUs in the middle SCS. Their distribution along depth shows increasing diversity toward the deeper sites. Phylogenetic inferences recover S. polita closely related to S. alucia from the Southwest Atlantic, forming a lineage deeply divergent from others. The nine deep‐water species/OTUs are scattered in three distinct lineages showing closer phylogenetic relationships between 1604‐ and 3402‐m counterparts. The lineage formed by S. naihaiensis and S. feitiana is distinct from other non‐SCS congeners both morphologically and genetically. These results suggest multiple independent invasions of Sirsoe to the SCS, a new lineage potentially endemic to the SCS, and a strong zonation pattern related to depth, especially between the shallow (655 m) and the deep (1604 and 3402 m) sites. The semi‐enclosed feature combined with the physical structure of the SCS may contribute to such a pattern. This work is registered in ZooBank under: urn:lsid:zoobank.org:pub:317771C8‐42D717‐4765‐A168‐B3BE99B09FBF.


| INTRODUC TI ON
As the largest marginal sea in the tropics, the South China Sea (SCS) has a semi-enclosed basin bordered by the Asian continent to the north and west, and separated from the West Pacific by Taiwan, Philippine Islands, and the Greater Sunda Islands. Water exchanges between the SCS and its surrounds are mainly through the Luzon Strait (LS), where a vertical sandwich-like inflow-outflow-inflow is formed in the upper-middle-deep layers. Water transport through LS is much stronger in the upper layer than in the middle layer. The deep layer lacks direct water exchange due to an isolated basin of deep SCS which is prevented by the sill in the LS (~2400 m) (Cai et al., 2020;Gan et al., 2022). In the context of its geologically semi-enclosed nature and strongly stratified physical structure, we hypothesize that the deep SCS harbors benthic invertebrate with a high level of endemism and depth zonation pattern, as what is observed in the Mediterranean Sea (Danovaro et al., 2010), and lineages deeply divergent from relatives outside. And a taxa group with members colonizing different depths can be used to test these hypotheses.
So far, little is known about the deep-sea biodiversity in the SCS, except for cold-seep fauna and cold-water corals from a few sites Li & Wang, 2019;Zhao et al., 2020). However, they cannot be used to test these hypotheses due to a limited depth range of the locations and/or a lack of genetic data. Recently, a small cetacean fall has been reported from a SCS seamount , evidencing the existence of such kind of habitats in the region for the first time despite their worldwide distribution (Smith et al., 2015). Investigation of cetacean falls usually recovers a wide variety of animals which can hardly be encountered when surveying the background environments (such as ocean basins and seamounts) and shows strong reliance on such habitats rich in nutrients and energy, thus revealing "hidden" biodiversity in a region (Smith et al., 2015). Although natural cetacean falls can only be encountered by chance, artificially implanted vertebrate carcasses, mimicking whale falls, at different depths provide alternative methods to study fauna associated with organic falls and an opportunity to test the hypothesis on the SCS biogeography (Amon et al., 2014(Amon et al., , 2017Braby et al., 2007;Fujiwara et al., 2007).
Animals of different nutrition modes emerge at cetacean falls in a successional sequence of stages: mobile-scavenger stage, enrichment-opportunist stage, sulfophilic stage, and reef stage (Smith et al., 2015). Among them, Sirsoe and Vrijenhoekia from the family Hesionidae are two important opportunists recovered from a wide variety of depth ranges (Pleijel et al., 2008;Shimabukuro et al., 2019). The two genera show high similarity to each other in both morphology and genetics, and were initially recovered as sister clades in early phylogeny (Rouse, Carvajal, et al., 2018;Summers et al., 2015). The main morphological characters distinguishing them were attributed to the absence of a median antenna, the presence of glandular lip pads (GLP), and well-developed neuropodia starting on segment 4 in Vrijenhoekia rather than Sirsoe (Pleijel et al., 2008).
However, this diagnosis was challenged by subsequent discoveries of Vrijenhoekia members with median antenna and paraphyletic status of those diagnostic features in a phylogeny with wider taxa sampling (Shimabukuro et al., 2019). Thus, Shimabukuro et al. (2019) adapted the diagnosis of Sirsoe to accommodate members in Vrijenhoekia and synonymized the two genera, resulting in a monophyletic Sirsoe, which is accepted in the present study.
Currently, Sirsoe species are mainly reported from whale falls from the Northeast (NE) Pacific and the Southwest (SW) Atlantic close to the Brazilian coast (Pleijel et al., 2008;Rouse, Carvajal, et al., 2018;Shimabukuro et al., 2019;Summers et al., 2015), and scattered records are also from hydrothermal vents and cold seeps from the Gulf of Mexico, off Costa Rica coast, Caribbean Sea, Okinawa Trough, and Mariana Trough (Blake, 1991;Desbruyères & Toulmond, 1998;Pleijel, 1998;Rouse, Carvajal, et al., 2018;Rouse, Goffredi, et al., 2018). A phylogenetic study revealed three clades in this genus, all of which, however, did not cluster by neither zoogeographic zones nor habitat types, except for three seep species forming a subclade in clade I (Shimabukuro et al., 2019). Most species seemed to be locally distributed, while inter-basin distribution was also confirmed in S. balaenophila "stricto sensu" and S. siriko, and depth might play an important role in diversification between S. alphacrucis and S. yokosuka (Shimabukuro et al., 2019). However, the western Pacific species were absent from available phylogenetic studies due to a lack in molecular data, leaving large gaps in knowledge of their biogeography.
Recently, we found abundant Sirsoe worms associated with implanted cow falls at three depths on Zhongnan seamount in the SCS. Here, we combine the specimen examination, phylogenetic and metabarcoding analyses to (1) describe and characterize the worms

| Sample collection and preservation
Zhongnan seamount is a conical seamount located in the middle SCS basin, with a depth range from 288 m at the top to 4355 m at the bottom ( Figure 1). In March 2021, three cows (with all internal organs removed, about 800 kg on average) were deployed at separate

T A X O N O M Y C L A S S I F I C A T I O N
Biodiversity ecology, Biogeography, Phylogenetics, Taxonomy depths on Zhongnan seamount in the SCS using a remotely operated deployer system during the TS2-5 cruise of R/V Tansuo2.
We revisited the deployed cows with the manned submersible Shenhaiyongshi, during the TS2-8 cruise of R/V Tansuo2 in July 2021, about 4 months after the deployment. Dense aggregations of benthic animals were observed. Polychaete worms were collected using either a suction sampler or pushcore. Sampling information is detailed in Table 1. Most samples were fixed and preserved in ethanol, with a few frozen separately under −20°C.

| Specimen repositories
Type specimens and material examined are deposited at the Repository of the Second Institute of Oceanography (RSIO), Ministry of Natural Resources, Hangzhou, China.

| Morphology
Specimens are morphologically examined under a stereomicroscope (Zeiss Discovery V.16). Optical images are taken using a CCD camera mounted on the stereomicroscope. To show details of neurochaetae, scanning electron microscopic (SEM) images are obtained following methods described in Han et al. (2021).

| Detection of Sirsoe with metabarcoding analyses
To examine if any other Sirsoe species in addition to the sampled specimens is present at each location, a surface layer (0-4 cm) of sediment is collected using pushcore sampler from each of the three cow falls. Only one sediment sample was obtained from each of the 655-and 1604-m site, while two separate sediment samples were collected at the 3402 m, both of which are treated as reciprocal "biological replicates" to address the concern about amplification errors and confirm the presence of each detected OTU at this site. For each sample, about 2-5 g sediment is used for total DNA extraction and then prepared for subsequent metabarcoding sequencing. Short fragments of COI are amplified using primers mlCOIintF (5′-GGWAC WGG WTG AAW ACW GGW TGA ACCYCC-3′) (Leray et al., 2013) and jgHCO2198 (5′-TAIAC YTC IGG RTG ICC RAA RAAYCA-3′) (Geller et al., 2013). The Illumina high-throughput sequencing of amplicons is performed on Illumina NovaSeq platform at Mingke Biotechnology Co., Ltd. Raw data filtration is performed using Trimmomatic v0.33 and cutadapt 1.9.1, and the resulting high-quality reads are assembled with FLASH v1.2.7 (Magoč & Salzberg, 2011) and then processed in UCHIME v4.2 (Edgar et al., 2011) to remove chimeric sequences. As 3% divergence of COI is the threshold value for inter-OTU delineation commonly accepted in metabarcoding research , and also the lowest value of COI divergence observed between nominal Sirsoe species (see below in the "Molecular and phylogenetic analyses"), sequences with ≥3% divergence from each other and nominal species are assigned to distinct OTUs using USEARCH (version 10 http://drive5.com/upars e/). The OTUs, which represent less than 0.01% of the total reads, were excluded in subsequent analyses except when they are present in both replicates.  Compared with the relatively high inter-lineage K2P distances (3.3%-29.6%) (  Sirsoe methanicola (Desbruyères & Toulmond, 1998)   show genetic divergence at the lower range of interspecific distance (K2P distance 3.3%-4.7%) (Figure 3).
Therefore, the suggested formal Chinese name is "光洁神女虫".
Remarks. Sirsoe polita sp. nov. resembles S. alucia in most characters, except for the proboscis, which bears significant ciliation between terminal papillae in the latter species. The two species also have a COI divergence (3%-4%) with each other much lower than with other congeners, which is the lowest interspecific COI divergence value in this genus. Parapodia sub-biramous (Figure 5e-g). Notopodia reduced and fused with dorsal cirriphore, bearing 1-2 acicula (Figure 5e-g).  Neuropodia well-developed, with triangular prechaetal acicular lobes and shorter rounded postchaetal lobes; dorsal projections on neuropodia pointed, much longer than prechaetal acicular lobes (Figure 5e-g).

Distribution. Currently
Parapodia sub-biramous, with triangular prechaetal acicular lobes, shorter rounded postchaetal lobes and very long pointed dorsal projections (Figure 6d,e). Neurochaetae numerous, compound, forming fan-shaped bundles, with supra-acicular neurochaetae longer than sub-acicular ones (Figure 6d,g). Supra-acicular and subacicular neurochaetae with shorter blades than middle ones. Blades distally curved, unidentate, with sub-distal prolongation and finely serrated cutting edges (Figure 6g,h). Pygidial cirri one pair, slender and terminal (Figure 6f). weight, and amount of bone lipids), we doubt that if differences in substrate type (cows vs. whales) can be evoked to explain such variations in hesionid diversity. Rouse, Goffredi, et al. (2018)  The failure of sampling of the OTUs detected by metabarcoding may be due to either their low abundance present in the community, or high heterogeneity at a fine temporal-spatial scale (Rouse, Goffredi, et al., 2018;Smith & Baco, 2003;Smith et al., 2015). in the Mediterranean Sea (Danovaro et al., 2010). The LS has been proposed as a boundary between the SCS and neighboring provinces (Spalding et al., 2007). Its barrier effects on population connectivity have been uncovered in several seep invertebrates, which show clear genetic break across the strait (Shen et al., 2016;Xiao et al., 2020;Xu et al., 2018Xu et al., , 2021  ) are attributed to their long-distance dispersal capability, deduced from an elongated planktonic larval stage of these species (Rouse et al., 2009) or their relatives (Eckelbarger et al., 2001), and a relatively small distance between suitable substrates in the form of vertebrate carcasses, which are presumably in sufficient supply by widespread marine vertebrates (Rouse, Goffredi, et al., 2018 This zonation pattern of Sirsoe may be partially explained by physical oceanography in the SCS. As the SCS is a semi-enclosed marginal sea, water exchange between the SCS and surrounding seas is mainly driven by a three-dimensional circulation, while the LS is the key connection between the SCS and the western Pacific Ocean. The upper layer inflow (<750 m) of LS was induced by wind stress and westward intrusion of the Kuroshio Current, the middle layer outflow (750-1500 m) and deep layer inflow (>1500 m) were influenced by the topographic effects and interior dynamical adjustment (Cai et al., 2020;Gan et al., 2022). The vertical inflow-outflow-inflow through the LS is one of the main driving forces inducing a cyclonicanticyclonic-cyclonic circulation of the SCS, and consequently results in the formation of water masses governing distinct depth range and weak vertical mixing except for areas over the slope in the north and south, where topography-current interaction invokes intensified mixing (Cai et al., 2020). At a finer vertical scale, two water masses exist below 1000 m, with the boundary at about 2700 m (Fengqi et al., 2002). According to this physical scheme, the three deployed cow falls, from shallow to deep, are located in three distinct water masses, the Intermediate Water Mass, the Deep Water Mass and the Bottom Water Mass (Fengqi et al., 2002), respectively. Combined with the phylogenetic structure, we speculate that colonization of Sirsoe speices to the upper (<750 m) and deep layer (>1000 m) depth range might be induced via distinct water mass and the strong depthrelated gradient between the two layers may act as dispersal barrier preventing vertical admixture of them. And the water mass subdivision below the 1000 m may also contribute to the differentiation between the 1604-and 3402-m cow falls.

Distribution. Currently
Biotic factors may also contribute to the zonation pattern.
One notable phenomenon observed during our investigations of the three cow falls was that the 655-m site was visually dominated by another worm in the family Chrysopetalidae compared with the two deeper sites, where Sirsoe speices predominated the communities (Zhou, Y., Xie, W., Yin, K., & Zhang, D., unpublished data). Although in need of further evidence, we speculate that Sirsoe may either prefer deep-sea environments, or be outcompeted by other more competent invertebrates in shallower water environments. Co-occurrence of multiple Sirosoe species/OTUs at the two deep sites might be attributed to either niche partition in food, space or time between relatives, mechanism of which has got supports from studies on isotopic diet analyses and been used to explain the coexistence of relatives observed in hesinoids and dorvilleids (Alfaro-Lucas et al., 2018;Thornhill et al., 2012). In addition, the temporal large amount of food (represented by mammal carcasses) can support high number of individuals feeding on them and may consequently allow coexistence of more species (Worm & Tittensor, 2018).

ACK N OWLED G M ENTS
We thank the captain and crews of R/V Tan Suo 2. We also thank the pilots and technical team of the HOV Shenhaiyongshi for their help in sample collection during the dives. We are grateful to Prof. PENG Xiaotong, Prof. YIN Kedong and Prof. YU Kefu for their support during the cruises.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
DNA sequences: GenBank accession numbers provided in Tables 2   and 3.